Development of a small‐scale bioreactor: Application to in vivo NMR measurement

A perfused bioreactor allowing in vivo NMR measurement was developed and validated for Eschscholtzia californica cells. The bioreactor was made of a 10‐mm NMR tube. NMR measurement of the signal‐to‐noise ratio was optimized using a sedimented compact bed of cells that were retained in the bioreactor by a supporting filter. Liquid medium flow through the cell bed was characterized from a mass balance on oxygen and a dispersive hydrodynamic model. Cell bed oxygen demand for 4 h perfusion required a minimal medium flow rate of 0.8 mL/min. Residence time distribution assays at 0.8–2.6 mL/min suggest that the cells are subjected to a uniform nutrient environment along the cell bed. Cell integrity was maintained for all culture conditions since the release of intracellular esterases was not significant even after 4 h of perfusion. In vivo NMR was performed for 31 P NMR and the spectrum can be recorded after only 10 min of spectral accumulation (500 scans) with peaks identified as G‐6P, F‐6P, cytoplasmic Pi, vacuolar Pi, ATP γ and ADP β , ATP α and ADP α , NADP and NDPG, NDPG and ATP β . Cell viability was shown to be maintained as 31 P chemical shifts were constant with time for all the identified nuclei, thus suggesting constant intracellular pH. © 2004 Wiley Periodicals, Inc.